Fig. 7. Di-leucine-based motifs are important for the intracellular localization and trafficking of syntaxins 7 and 8. A putative di-leucine-based motif is present in the cytoplasmic domains of syntaxin 7 (amino acids 162-168) and syntaxin 8 (amino acids 77-83). Mutations in these motifs were made as indicated by underlining. Clone 9 cells were transfected with syn7-mut (a,b) or syn8-mut (d,e), each of which was fused to an N-terminal HA tag. For intracellular localization analysis, the cells were incubated for 12 hours (a,d) or 24 hours (b,e) and stained with anti-HA antibody. For antibody uptake experiments, Clone 9 cells were transfected with mutants in which the C-termini were fused to three c-myc tags (c,f). After 24 hours incubation, the cells were incubated for 3 hours in the presence of mouse anti-c-myc antibody before fixing. The cells were fixed and stained with FITC-conjugated anti-mouse IgG. (a-c) Syn7-mut was localized to endosomes and at the plasma membrane. In antibody uptake experiments, labeling was found only at the plasma membrane. (d-f) Syn8-mut was localized to the perinuclear Golgi region. No staining was observed in antibody uptake experiments.